Cytosolic 43 KD protein of Amoeba proteus purified by DNase-1-affinity chromatography or partially purified by eluting from preparative SDS polyarcylamide gels, was characterized and compared with actins from other sources using monoclonal antibody against the 43 KD protein (Kim and Jeon, Endocyt. C. Res. 4: 151, 1987) as the probe. The monoclonal antibody cross-reacted with actins from skeletal muscles (bovine, porcine, rabbit, chick), non-skeletal muscle (rabbit heart, chicken-gizzard) and Acanthamoeba, but did not react with Naegleria action. On IEF gels the 43 KD protein resolved into 3 bands with pIs of 5.96, 6.03 and 6.10 which were in the range of typical profiles of vertebrate actins that shows pIs of 5.75, 5.80 and 6.0. The protein was resolved into 2 bands with pIs of 5.96 and 6.03 after treating with alkaline phosphatase. When the 43 KD protein was partially digested with chymotrypsin and analyzed by 2-D PAGE and immunoblotting its peptide map closely resembled those vertebrate actins having the most of the peptides in common, Thus the 43 KD protein of amoeba was confirmed as actin. The peptide map of amoeba actin after tryptic digestion was the more diverse than that of chymotryptic digest, although there were still many common peptide spots. The uniqueness of amoeba actin was most pronounced after digestion with V8 protease which cut the skeletal muscle and non-muscle actin into two pieces. The monoclonal antibody was found to cross-react with the C-terminus half of muscle actins after digesting with V8 protease.